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Submitted on June 28, 2004
Novartis Institute for Biomedical Research, Inc., Cambridge, MA 02139
Corresponding Author: karen.wang{at}pharma.novartis.com
Tyrosine phosphorylation is a type of post translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, due to the low abundance of tyrosine phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phospho-tyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phospho-peptides were further enriched by immobilized metal affinity chromatography (IMAC) and analyzed by liquid chromatography coupled to mass spectrometry (LC-MS). Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double-enrichment approach, we characterized interferon a (IFNa) induced phospho-tyrosine proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNa signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on a-tubulin (Y271) was observed to be phosphorylated upon treatment as well. Further more, our results suggest that LOC257106, a CDC42 GAP like protein, is potentially involved in this pathway.
Revised on January 18, 2005
Accepted on January 19, 2005
Phospho-tyrosine proteomic study of interferon asignaling pathway using a combination ofimmunoprecipitation and immobilized metal affinity chromatography
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